Phenotypic Characterization And Candidate Gene Discovery Of Internode Related Traits Of EHel With Extremely Low Ear Height In Maize (Zea Mays L.)

Plant architecture is closely related to canopy structure,lodging,photosynthetic efficiency and yield of maize.In breeding practice,reasonable plant height and ear height are important conditions to ensure stable and high yield of maize.The number and length of internodes are the basic components in determining the performance of plant height and ear height in maize.Up to now,only limited publications were focused on internode related traits,such as internode number and internode length.It is of great practical significance to further analyze the genetic mechanism of plant height and ear height from the internode characters of maize.In the present study,EHel was crossed with B73,to construct segregating populations of F₂ and F_2:3.10K-SNP chip was used to scan the population with high density of genome,and the polymorphic SNP construction linkage map was screened out.QTL mapping was carried out based on the phenotypic characterization of internode related traits.At the same time,combined with the sequencing data of B73,the main QTL loci were annotated and candidate genes were screened,EHel and B73 were used to analyze the expression and sequence of the identified candidate genes to carry out the preliminary verification of the candidate genes.The main results are as follows:1.Phenotypic characterization of EHel internode related traitsThe phenotype analysis results of internode correlation traits showed that EHel showed extremely significant differences in plant height,ear height and EH/PH with B73and F₁.The difference in the average internode numbers above ear between EHel and B73is inconsistent between two years.However,the average internode numbers below ear,average internode length below ear,and average internode length above ear were significantly lower than B73 between 2018 and 2019.In addition,the statistical analysis results also show that seven target traits of F_2:3 family all conform to the Gaussian distribution.2.Correlation analysis of study traitsThe correlation analysis results of the target traits showed that the plant height,ear height,ratio of ear height to plant height,internode numbers below ear,average internode length above ear and average internode length below ear all showed extremely significant positive correlation?P<0.001?.Among them,the correlation coefficient with average internode length below ear is as high as 0.8;the correlation coefficient with the ear height is 0.79;the correlation coefficients with the EH/PH,internode numbers below ear,and average internode length below ear are 0.58,0.59,and 0.57,respectively.The correlation between ear height,internode numbers below ear and average internode length are also high,with the correlation coefficients reaching 0.82 and 0.78 respectively.3.Linkage map constructionMaize 10K SNP chip was used to determine the genotypes and polymorphisms of the parents and F₂ populations to construct linkage map.Two thousand one hundred and eight polymorphic SNPs were screened from?10000,the total length of the map was3299.18 cM,and the average spacing of SNP markers was 1.57 cM.On 10 chromosomes?Chr?,the number of polymorphic SNP markers on five chromosomes such as Chr1,Chr3,Chr5,Chr7,Chr8 were all greater than 300,which was relatively large.From the analysis of the average marker spacing of each chromosome,the average marker spacing of five chromosomes,such as Chr1?1.11 cM?,Chr2?1.19 cM?,Chr4?1.38 cM?,Chr6?1.33 cM?,Chr10?1.13 cM?,was lower than Genome-wide average marker spacing.4.QTL discovery of target traitsIntegrating the phenotype of F_2:3 strain,the QTL mapping of target traits was carried out using the composite interval mapping method,and a total of 22 QTL loci were identified.Among them,three QTLs controlling plant height were identified on chromosomes 2,4 and 6;three QTLs controlling ear height and EH/PH were identified on chromosomes 1,2,and 6;four QTLs controlling internode number above ear were identified on chromosomes 2,7,and 8.Three QTLs with an average internode length above ear were identified on chromosomes 1,2,and 5,qILaa5 was the main QTL.Only one QTL related to the internode number below ear was identified on chromosome 8;five QTLs controlling the average internode length below ear were detected on chromosome2 and 6.The phenotype variation rate of a single QTL is between 6.52%and 13.00%.qPH6,qEH6 and qILab6 on chromosome 6 are all located at the same position,which is the same QTL.From the perspective of their confidence range,qILaa1 and qILaa5 are relatively small,respectively 1 cM and 2 cM.5.Preliminary validation of candidate genesAccording to the QTL mapping results,it was found that the main effective QTL site qILaa5 located on chromosome 5 that controls the average internode length above ear has the largest LOD value among all the QTL sites located,and the phenotype variation rate reaches 12.99%.The size of the formed chromosome segment is 1.004 Mb.Systematic gene annotation and screening of this segment revealed that Zm00001d016823 and Zm00001d016831 may be candidate genes for controlling internode length.The results of qPCR analysis based on candidate gene showed that the relative expression levels of Zm00001d016823 and Zm00001d016831 in internodes were significantly higher than those in leaf sheaths,leaves,ear and roots.We design specific primers for candidate genes,amplify their full-length sequences,then clone and sequence candidate genes.Sequencing results found that there is no difference in the sequence of Zm00001d016831 in EHel and B73,Zm00001d016823 has 11 polymorphic sites in EHel,including two insertion/deletion?Insertion/Deletion,In Del?and nine polymorphic SNPs,all located in the coding region of the gene.Of the two Indel polymorphisms,one is 12 bp?TGATCCGGCGGC?deletion?InDel ??;another one is 6 bp?AGAAGA?deletion?InDel II?.